a master lecture for plant breeding about RFLP marker

1. RFLP Marker
By
Afnan Said Zuiter

2. Definition
• (often pronounced "rif lip", as if it were a word)
• RFLP - Restriction Fragment Length Polymorphism; a molecular
marker based on the differential hybridization of cloned DNA to
DNA fragments in a sample of restriction enzyme digested DNAs
(endonuclease); the marker is specific to a single clone/restriction
enzyme combination, and can be detected by Southern blot
(hybridization with a probe).

3. Polymorphism
• RFLPs have polymorphisms (length differences in homologous
fragments between different DNA) which caused by changes in the
primary sequence of DNA.
• Polymorphisms can be resulted of:
– Point mutation resulting in the loss or gain of a restriction
enzyme cut site.
– An insertion or deletion of DNA between two restriction
enzyme cut sites
– A deletion which overlaps a restriction enzyme site
– A DNA rearrangement where one end of the rearranged segment
resides between two restriction enzymes site.

4. Forensic Science: An Encyclopedia of History, Methods, and TechniquesForensic Science: An Encyclopedia of History, Methods, and Techniques
William JWilliam J.. Tilstone, Kathleen ATilstone, Kathleen A.. Savage, Leigh ASavage, Leigh A.. ClarkClark
Kathleen AKathleen A.. Savage, Leigh ASavage, Leigh A.. ClarkClark
ABCABC--CLIOCLIO, 2006, 2006
ISBNISBN 1576071944, 97815760719461576071944, 9781576071946
307307

5. Advantages
• They are codominant (both alleles in heterozygous sample will be
detected) and unaffected by the environment;
• Any source DNA can be used for the analysis; no prior informations
are needed
• Many markers can be mapped in a population that is not stressed by
the effects of phenotypic mutations.
• They don’t require high level of skill.
• They have high reliability.
• They have medium polymorphism, allelic information, and robustness

6. Special advantage (in plant molecular
breeding)
• The integration of RFLP techniques into plant breeding promises to:
(1) Expedite the movement of desirable genes among varieties,
• (2) Allow the transfer of novel genes from related wild species,
• (3) Make possible the analysis of complex polygenic characters as
ensembles of single Mendelian factors,
• (4) Establish genetic relationships between sexually incompatible
crop plants.
• (5) In the future, high density RFLP maps may also make it possible
to clone genes whose products are unknown, such as genes for
disease resistance or stress tolerance.
• S. D. Tanksley1, N. D. Young1, A. H. Paterson1 & M. W. Bonierbale1, 1989, RFLP Mapping in Plant
Breeding: New Tools for an Old Science (review), Bio/Technology 7, 257 - 264
doi:10.1038/nbt0389-257 (http://www.nature.com/nbt/journal/v7/n3/abs/nbt0389-257.html)

7. Disadvantages
• Only relatively low input can be achieved (Samples per day in the
research lab is low (20 sample)).
• Number of samples is moderately low (<300-400)
• Labor intensive.
• Developmental costs ($/sample) are medium (100)
• Running costs ($/sample) are high (2)
• Quantities of high quality DNA are required
• Gel based and thus not easily automable
• Requires relatively large amounts of high quality DNA

8. Applications
• The medium polymorphism, allelic information, and robustness are
very useful for 
1. mapping
2. Fingerprinting (paternity tests, criminals detection)
3. Population studies
4. Diversity classification
5. Phylogenetic studies ( if they are recorded as restriction site
data)

12. httphttp://://wwwwww..accessscienceaccessscience..comcom//conteconte
ntnt..aspx?idaspx?id==535500535500

13. Technique and Procedure

14. Isolate DNA to be analyzedIsolate DNA to be analyzed
Digest DNA with restriction enzymeDigest DNA with restriction enzyme
Perform agarose gel electrophoresisPerform agarose gel electrophoresis
Transfer DNA from gel to membraneTransfer DNA from gel to membrane
Hybridize with labeled probeHybridize with labeled probe
Wash membrane toWash membrane to
Remove unhybridize probeRemove unhybridize probe
AutoradiographyAutoradiography
Reuse ofReuse of
membranemembrane
RFLP analysisRFLP analysis

15. Equipments
• Horizontal agarose gel electrophoresis
• Oven for hybridization (60-65°C)
• Drying oven (80°C)
• Shaking incubator for post-hybridization membrane
• Rocker shaker for treating gels
• Fluorometer for DNA quantitation

16. Solutions
• NaCl + sodium citrate (pH=7, RT)
• SSPE (NaCl + Na2HPO4 + EDTA) pH=7.4 , RT
• TAE (Tris base + Glacial acetic acid + EDTA)
• Gel loading mix
– Formulation A (Ficol + Tris-HCL + Bromophenol blue)
– Formulation B (Glycerol + H2O + bromophenol blue
• Denhardt’s solution (Ficol + polyvinylpyrolidone + Bovin serum albumin +
H2O (4oC)
• NaP i solution (monobasic + dibasic sodium phosphate) pH=6.7
• Hybridization solution (dH2O + SSC + SDS + NaPi) + Denhardt’s solution
+ sample
• OLB (offset length buffer)
– Solution O: Tris-HCl + MgCl2
– Solution A: solution O + dNTP
– Solution B: HEPES pH=6.6
– Solution C: Ud(N)6 + Tris-HCl + EDTA

17. (Isolation + Digestion)
1. Prepare the DNA sample (DNA isolation), usually by a strategy based
on the use of the cationic detergent cetyl-trimethylammonium
bromide (CTAB) for DNA precipitation
(http://www.ncbi.nlm.nih.gov/pubmed/2699240)
• Genomic DNA CTAB method (1-2 ug per lane)
• Plasmid DNA  minipreparation (0.1-0.5ug per lane)
2. Verify the relative concentrations of all DNA samples
3. Restriction enzyme digestion (1-2 U/ug DNA) in 25ul volume, 4h, RT
add water, buffer, DNA, then the enzyme
sometimes we need more than one enzyme to digest (so need to
sequential digestion with ethanol precipitation between them in order
to change salt concentration)
add gel loading buffer to:
• Stop enzymatic reaction, and dissociation the enzyme from
DNA
• Add a tracking dye to monitor electrophoresis
• Increase the density of the sample to stay in the well

18. Experimental Protocol (gel electrophoresis)
• It is agarose gel electrophoresis
• Horizontal
• 7.5 cm of gel down-current of the comb
• 0.8-1% agarose, 250ml
• TAE buffer
• 20x25 cm gel tray
• 30V for 18-20 h
• Using DNA marker for genomic DNA (HindIII-digested phage
lambda DNA)
– 1ug/lane  to be visualized using EtBr  302 nm UV
» In gel (0.5 ug/ml)
» Post electrophoresis staining
– 300ng/lane  to be visualized by autoradiography

19. (Minnesota Molecular,(Minnesota Molecular,
Inc., USA)Inc., USA)

20. Digested DNA withDigested DNA with XalXal Undigested genomic DNAUndigested genomic DNA

22. (Blotting)
• The transfer of the DNA from the gel to a suitable membrane where it is ready to
hybridization with a probe.
1. Cut of the gel above the wells and bellow the bottom of DNA smear
2. Conduct the following steps in a glass or plastic tray on a shaker at room
temperature
1. Depurinate DNA in 0.25 M HCl for 7min (bromophenol blue yellow)
2. Rinse with dH2O for 15-30s
3. Denature DNA in (NaCl +NaOH) for 10-15min (bromophenol blue blue)
4. Rinse with water for 15-30s
5. Raise salt concentration with 1.5M NaCl for 5 min
3. Southern transfer:
• the stack is assembled on a stage piece of plastic wrap (completely
enclosed to keep gel & membrane from drying out)
• 2-3 dry paper towels act as a sink of the liquid being transferred by
capillary action from the gel
• Nitrocellulose or nylon membrane is used for DNA transfer

23. Blotting
• The gel is placed on the membrane right side up and centered so that
there is a border of membrane all around the gel.
• All bubbles should be removed (DNA will not take place through it)
• Incubate overnight at room temperature –or- 1 h by placing a sponge
soaked in 10X SSC on top of the gel instead of the weight.
• Disassemble blotting stack and wash the membrane in 5X SSC for 5-10
min –or- 0.1M Tris HCl (pH=7) + 1M NaCl for 15min shaking, RT
• Remove membrane and quickly blot dry using clean paper towels.
4. Fix DNA to the membrane:
• Place the damp membrane between paper towels
• Bake it at 80oC for at least 1h

24. httphttp://://wwwwww..koreanbiokoreanbio..orgorg//BiocourseBiocourse//indexindex..phpphp//HybridizationHybridization

25. Experimental Protocol (Probe labeling)
A. PCR amplification of plasmid insert  to prepare a probe for labeling
1. Add the reagents (DNA + Taq Pol. + MgCl2 + dNTPs + Primeres + H2O)
2. Amplify DNA by specific thermal cycle profile
3. A 1% agarose minigel can be used to check the success of the
amplification and to estimate the yield of amplified product
A. Labeling of probe prior to hybridization (nick translation, end labeling,
random priming)
• Add these reagents:
1. 50-100ng PCR reaction product
2. H2O  incubate at 100oC for 3-5min and quick chill on ice.
3. OLB
4. BSA
5. 32P-dCTP
6. Klenow fragment of DNA pol I.
• Incubate at RT for 3-4 h

26. Hybridization
A. Prehybridization
– Prehybridize new membranes for 3h to overnight at 65oC
– It is done in plastic bottles on a rotator in a convection oven –or-
using a shaker (the solution and the probe should keep in constant
movement over the membrane to ensure an even signal)
– Multiple membranes can be hybridize in a single bottle
 15ml for 1-2 membranes
 25-30ml for 3-6 membranes
**it is important to not let the membranes to dry
**also keeping the solution to the minimum (to have high probe conc.
and reduce the amount of radioactive waste that must be discarded)
B. Hybridization:
1. Denature the probe by heating to 98-100oC for 5 min
2. Quick chill on ice
3. Add probe to the prehybridization solution (in the bottle) & mix
4. Incubate at 65oC in an oven for 16-20h -or- 48h in low probe
con.

27. Post hybridization washes
• To remove all the nonspecifically bound probes.
• Controlling membrane background and signal intensity
• All solutions should be preheated
1. Low stringency wash
Incubate the membrane in a tray and shake gently at 60-65oC for 15-
20min using SSC and SDS solutions.
2. High stringency wash
– wash using SSC and SDS solutions at 60-63oCfor 15-20min
– Test the efficacy of non-hybridized probe removal with a Geiger counter
– <2000 cpm are needed for good quality autoradiograms
If the membrane background is still high:
1. Repeat the high stringency wash with fresh solution
2. Increase the incubation temperatureto 65oC
3. Lower the salt concentration of the wash solution to 0.1 SSC, 0.1 SDS
– Now the membrane is quickly blotted dry with paper towels in a seal-meal bag
 to not permanently fix the probe to the membrane and prevent its reuse.

28. Autoradiography
• Should be performed at -70oC
• Exposures typically range from a few hours to 7 days
• Longer exposures are helpful in detecting weak signals
• Intensifying screens convert the energy of beta particles to photon
which can then enhance exposure of the film.

29. Reusage of membranes
• To hybridize the membrane with several different probes
• The first probe must be removed before reuse using pH
– Strip probe with NaOH , SDS solution for 5 min
– Wash with 2 quick rinses of dH2O
– Restore high salt in 5X SSC solution for 20min
• Use 50-100ml of solution per membrane
• All steps are done with shaking in a tray at RT

30. Conclusion
• Using RFLP is very useful in genetic analysis where the number of
samples is moderately low (<300-400)
• If many samples must be analyzed or information of many markers
is needed, it may be appropriate to use one of the amplification –
based techniques that have been recently developed.

31. • http://books.google.jo/books?
id=zIRQOssWbaoC&pg=PA48&lpg=PA48&dq=1980+-
+Ray+White+describes+first+polymorphicRFLP+marker+
%2B+pdf&source=bl&ots=wF-1uYXR0Q&sig=qv-clgqKH--
Nnlwaq2URrIWrEr0&hl=ar&ei=Y8GhScfAM5DDjAeEsq3DCw&s
a=X&oi=book_result&resnum=3&ct=result#PPA49,M1